The enzymes regulating the levels of cyclic nucleotides in cells govern some of the most important biological control systems, yet very little is known concerning the nature of their active sites or the detailed catalytic steps in the conversion of the nucleoside triphosphates to the cyclic nucleotides, and their subsequent hydrolysis. On occasion, this lack of knowledge contributes to a tendency to regard every behavior of these enzymes as manifests of complex molecular control schemes, when such behavior may, in actuality be due simply to normal kinetic behavior. In previous years, only grossly contaminated enzyme preparations were available for kinetic study, adding further complexity to the kinetic issue; however, methods for obtaining highly purified preparations of these enzymes are now becoming available. The proposed research details studies utilizing the classical techniques of enzymology to define the shape and binding properties of the active sites of highly purified preparations of adenylate cyclase, guanylate cyclase, and the cyclic AMP and cyclic GMP phosphodiesterases. Studies are described which hopefully will reveal the essential functional groups in the active site which actually perform the catalysis, and the step-by-step progress of the catalytic events involved in converting the substrate to product for each of the four enzymes. Classical enzymological techniques which will be used include turn-over kinetic studies, direct and in the presence of products, product analogues, and "transition state analogues"; stable isotope studies utilizing labeling of crucial substrate portions, kinetic isotope effects, and isotope exchange; substrate analogue kinetic studies; group specific reagents as probes for specific functional groups; and, the use of laser-Raman and/or electron paramagnetic resonance studies to define the nature of metal ion interactions with enzymes and substrates. The resulting complete kinetic description of each enzyme will provide a firm foundation for our ongoing project of designing differential kinetic inhibitors capable of selectively manipulating cellular levels of cyclic AMP and cyclic GMP, allowing eventual control over the important biological processes they regulate.